[Pathology didactics : Development and pilot study of a pathology didactics curriculum (PaDiCu)]. / Pathologiedidaktik : Entwicklung und Pilotierung eines Pathologiedidaktikcurriculums (PaDiCu).

BACKGROUND: Pathologists are lifelong teachers. However, specialist training contains hardly any didactic learning objectives. Here, the competency-based learning objectives that a pathology didactics curriculum could include were examined. MATERIALS AND METHODS: Learning objectives were determined through expert discussions and literature research. Four teaching units were designed: A) Interactive small-group seminars; B) The timely application of what has been learned under supervision; C) A longitudinal component by providing digital resources; D) Workshops and seminars by third-party providers. RESULTS: Initially, 11 small group seminars were designed: 1. + 2. General and special presentation techniques, 3. Public speaking, 4. Activation of audience, 5. Macro- and microphotography, image processing and graphic design, 6. Pathology teaching concept, 7. Standardized case conferences, 8. Standards for teaching events, 9. Standardized evaluation, 10. Research of pathology education, 11. Digitalization of teaching and pathology. Pilot seminars were appreciated and positively evaluated. CONCLUSION: For the first time, competence-based didactic learning objectives were identified for pathology. The pathology didactics curriculum (PaDiCu) could be disseminated with little effort.

[Treatability of sporadic late onset nemaline myopathy]. / Behandelbarkeit der "sporadic late onset nemaline myopathy".

Sporadic late onset nemaline myopathy (SLONM) is an extremely rare disorder which can be associated with monoclonal gammopathy of unclear significance (MGUS). Clinically SLONM appears mostly after the fourth decade of life as rapidly progressing tetraparesis, respiratory insufficiency and features, such as dropped head syndrome, facial and bulbar involvement. Diagnosis is confirmed by muscle biopsy with detection of nemaline bodies and also frequently lobulated fibres. Immunosuppressant and immunomodulating therapies have been shown to be ineffective but clinical improvement accompanied by disappearance of monoclonal gammopathy and even nemaline bodies was reported following autologous stem cell transplantation and chemotherapy with melphalan. This article presents the case of a 53-year-old man with a 4-year history of SLOMN with MGUS in which administration of intravenous immunoglobulin therapy (IVIG) was not successful in reversing gammopathy, histopathological changes or clinical symptoms.

Diagnostic utility of IDH1- and p53-mutation analysis in secondary gliosarcoma.

We report on a 47-year-old woman in whom an anaplastic astrocytoma was resected in 2006. Postoperative radiation had to be interrupted because of a wound infection necessitating explantation of the infected bone flap and implantation of a titanium mesh. Subsequently, radiation therapy was completed and temozolomide was administered for 45 cycles. In the beginning of 2010 a new contrast enhancing mass was seen in the former tumor region. The mass was subtotally excised and showed no histomorphological similarity to the first lesion but represented a highly pleomorphic and mainly sarcomatoid differentiated malignant tumor. The lack of expression of GFAP or MAP-2 raised the question of a secondary malignancy, however, molecular genetic analysis of IDH1 and p53 revealed the same mutations in the anaplastic astrocytoma from 2006 as in the sarcomatoid tumor operated in 2010. Furthermore, accumulation of mutated IDH1 and TP53 protein could be demonstrated immunohistochemically. Thus, the second tumor represented the rare instance of recurrence of an anaplastic astrocytoma as a secondary gliosarcoma and a second malignant neoplasm was ruled out. The postoperative therapy and the inflammation might have contributed to the severe change in morphological phenotype of the glioma.

Persistent toxoplasma bradyzoite cysts in the brain: incidental finding in an immunocompetent patient without evidence of a toxoplasmosis.

We report on a 72-year-old patient in whom autopsy demonstrated incidentally intracerebral Toxoplasma gondii cysts, locally restricted in the occipital lobe, in association with only a few CD4+ and CD8+ T cells and a mild microglial activation. The patient was HIV-negative. Serologically, there was no evidence for an active inflammation (Toxoplasma gondii specific antibody IgG-positive, IgM-negative). This unusual observation may indicate that in patients with sepsis, who may yield to a state of immunodysbalance, a focal reactivation of parasites may ensue in the absence of conditions predisposing for opportunistic infection.

Precursor T-lymphoblastic lymphoma within a recurrent pituitary adenoma.

Only single examples of lymphoma associated with pituitary adenoma have been reported. In our patient, a precursor T-lymphoblastic lymphoma developed within a recurrent pituitary adenoma 17 years after the first resection. Histomorphologically, lymphoma and adenoma components were tightly admixed. The features harbour remarkable similarity to the previous report by Kuhn et al.. In both patients the lymphomas were composed of T-cells, there was no evidence of further sites involved, and both adenomas expressed follicle-stimulating hormone. The hormone may have posed a proliferative and transforming effect on lymphatic cells and could have played a crucial role in "lymphomagenesis" as an exceptional phenomenon.

Proximal chromosome 11p contiguous gene deletion syndrome phenotype: case report and review of the literature.

The proximal chromosome 11p contiguous gene deletion syndrome (P11pDS), also known as Potocki-Shaffer syndrome (PSS) or DEFECT 11 (OMIM 601224), is a disorder associated with foramina parietalia permagna and multiple osteochondroma (exostoses). Additional features include mental retardation, craniofacial anomalies, seizures and genitourinary abnormalities. Here, clinico-pathological findings of a unique patient with all of these features and, additionally, enlarged ventricles, hypertrophic obstructive cardiomyopathy and adipositas are described. The brain showed malformative lesions with hallmarks of disturbed bulk growth including micrencephaly, periventricular nodular heterotopias and focal cortical dysplasia in the nodulus of the cerebellar vermis. In addition, symmetric foci with vacuolation of the underlying neuropil, intermingled macrophages and large bizarre, partially vacuolated, reactive astrocytes were found. The proximal short arm of chromosome 11 harbors several candidate genes that could explain the patient's signs and symptoms including ALX4 and EXT2, which are always present in the interstitial deletion of the short arm of chromosome 11 in PSS. In addition, MYBPC3 would be a good candidate for the hypertrophic cardiomyopathy. Furthermore, adipositas might be related to the MAPK8IP1 gene. To the best of our knowledge, the present patient is the oldest one so far described with PSS phenotype and the only case that has undergone detailed neuropathological investigation.

Automated nuclear segmentation in the determination of the Ki-67 labeling index in meningiomas.

OBJECTIVE: Assessing the Ki-67 labeling index (LI) is laborious and time consuming. Therefore, an automated computer-based method was developed, which is able to identify and analyze immunolabeled and hematoxylin-stained nuclei in digital images of routine immunohistochemical slides. MATERIAL AND METHODS: The method is based on a plugin for the public domain image analysis software ImageJ, which runs on every operating system (free download at http://rsb.info.nih.gov/ij/). Percentage of Ki-67 immunostained nuclei were determined in 5 high power fields (x40) of immunostained slides (DAB detection technique, hematoxylin counterstain) of 20 Grade I, 20 Grade II, and 10 Grade III meningiomas conventionally by two independent investigators and automatically, respectively. The time effort was measured for each counting procedure. RESULTS: Enumerating conventionally or automatically did not reveal any significant differences in the mean labeling indices. Ki-67 LIs discriminated sufficiently between meningiomas of Grade I (median 1.7% Investigator 1 and 1.5% Investigator 2 vs. 1.5% automatically), Grade II (7.6%, 8% vs. 7.3%), and Grade III meningiomas (22%, 21% vs. 22%). The computer-based results correlated very closely with those obtained by manual counting (correlation coefficient = 0.98). The mean time effort for counting procedure per image was 374 s (130 s-435 s) for the conventional and 11 s (7 s-12 s) for the automated method. CONCLUSIONS: The described method can reliably assess the Ki-67 LI much faster than conventional enumerating. The computerized method has the advantages of objectivity, accuracy, repeatability, and ease of use. There is no request for special stains nor special image acquiring systems. The plugin can be downloaded at the "Morphometrie" section of http://www.uniklinikum-saarland.de/neuropathologie.

Expression, cellular distribution and protein binding of the glioma amplified sequence (GAS41), a highly conserved putative transcription factor.

The glioma amplified sequence 41 (GAS41) was previously isolated by microdissection mediated cDNA capture from the glioblastoma multiforme cell line TX3868 and shown to be frequently amplified in human gliomas. We determined the complete cDNA sequence of the GAS41 gene, demonstrated that the GAS41 protein is evolutionarily conserved, specifically at the N-terminus, and identified the yeast transcription factor tf2f domain within the GAS41 sequence. A human multiple-tissue Northern blot revealed ubiquitous expression of GAS41 with the highest expression in human brain. After generating polyclonal antibodies we found GAS41 protein expression in the nucleus of the TX3868 cell line by Western blot analysis and immunofluorescence microscopy. The nuclear localization was confirmed for several human tumors including gliomas of different grades of malignancy. In neuroblastoma however, GAS41 was found in the nucleoli but not in the nucleoplasm. Yeast two-hybrid screening of the TX3868 cell line identified the nuclear mitotic apparatus protein (NuMA), the KIAA1009 protein, and prefoldin subunit 1 (PFDN1) as potential interacting partners of GAS41. We generated a polyclonal antibody against the KIAA1009 protein and we demonstrated that the KIAA1009 protein is a nuclear protein, which appears to be co-localized with the GAS41 protein and NuMA.

PHF3-specific antibody responses in over 60% of patients with glioblastoma multiforme.

Glioblastoma multiforme (GBM), a malignant astrocytic tumour, represents the most frequent tumour of the human brain. Nevertheless, its molecular pathology is not well understood. We utilized the immune system, which contributes to cancer protection, to help identify new GBM-related genes. By screening a human GBM cDNA library with autologous patient serum (SEREX-approach), we isolated a gene termed PHF3 (PHD finger protein 3). The gene product of PHF3 is immunogenic in GBM as tested in an allogenic patient serum screening demonstrating antibodies in 24 of 39 (61.53%) sera, whereas none of the 14 healthy persons had antibodies against PHF3. While previous SEREX studies revealed allogenic antibody responses up to 40%, our results for PHF3 represent the highest reported rate for a specific antibody response. We show that GBM patients with an antibody response against PHF3 show significant better survival than patients without PHF3-specific antibodies. Because the amino acid sequence of PHF3 contains a PHD finger (also termed LAP motif), a TFIIS homology, a proline rich region and nuclear localization signals, it supposedly functions as a transcription factor. A polyclonal antibody generated against PHF3 shows nuclear expression in most investigated formalin-fixed, paraffin embedded tissues. In GBM, PHF3 expression is concentrated in cells surrounding necroses.

Liquid-filled balloon brachytherapy using (68)Ga is effective and safe because of the short 68-minute half-life: results of a feasibility study in the porcine coronary overstretch model.

BACKGROUND: Liquid-filled balloons for coronary brachytherapy provide significant advantages over solid sources in dose homogeneity but carry the risk of life-threatening radiointoxication after balloon rupture and laboratory contamination in case of a spill. We hypothesized that the positron emitter (68)Ga, with a half-life of only 68 minutes, was well suited to overcome these safety obstacles while providing full therapeutic efficacy. METHODS AND RESULTS: The feasibility, efficacy, and safety of (68)Ga liquid-filled balloon brachytherapy were investigated in the porcine coronary overstretch model. Four groups of 5 balloon-induced coronary lesions were irradiated with 8, 12, 16, and 24 Gy targeted to the adventitia. Ten unirradiated lesions served as controls. Segments treated with 16 or 24 Gy exhibited marked suppression of neointimal proliferation at 28-day follow-up, with quantitative parameters of intraluminal proliferation reduced to <20%. This beneficial effect was not compromised by untoward edge effects. Uninjured but irradiated vessels did not show histological signs of radiation damage. The (68)Ga whole-body dose due to balloon rupture was estimated to be 5 rem/50 mCi treatment activity and compared favorably with that of (188)Re (78 rem/50 mCi). CONCLUSIONS: (68)Ga positron radiation suppresses neointimal proliferation at doses of 16 and 24 Gy. This biological efficacy, coupled with the attractive safety profile, suggests the selection of (68)Ga as an attractive isotope for liquid-filled balloon brachytherapy.

Expression of cancer testis genes in human brain tumors.

Cancer-testis (CT) genes are expressed in a variety of human cancers but not in normal tissues, except for testis tissue, and represent promising targets for immunotherapeutic and gene therapeutic approaches. Because little is known about their composite expression in human brain tumors, we investigated the expression of seven CT genes (MAGE-3, NY-ESO-1, HOM-MEL-40/SSX-2, SSX-1, SSX-4,HOM-TES-14/SCP-1, and HOM-TES-85) in 88 human brain tumor specimens. Meningiomas expressed only HOM-TES-14/SCP-1 (18% of meningiomas were HOM-TES-14/SCP-1 positive) and did not express any other CT genes. One ependymoma was negative for all CT genes tested. SSX-4 was the only CT gene expressed in oligodendrogliomas (2 of 5 cases), and it was also expressed in oligoastrocytomas (3 of 4 cases) and astrocytomas (10 of 37 cases). Astrocytomas were most frequently positive for HOM-TES-14/SCP-1 (40%) and SSX-4 (27%), followed by HOM-TES-85 (13%), SSX-2 (11%), and MAGE-3 (7%). Whereas MAGE-3 was detected only in grade IV astrocytomas, the expression of the other CT genes showed no clear correlation with histological grade. Of 39 astrocytomas, 60% expressed at least one CT gene, 21% expressed two CT genes, and 8% coexpressed three CT genes of the seven CT genes investigated. We conclude that a majority of oligoastrocytomas and astrocytomas might be amenable to specific immunotherapeutic interventions. However, the identification of additional tu-mor-specific antigens with a frequent expression in gliomas is warranted to allow for the development of widely applicable polyvalent glioma vaccines.

Immunohistochemical detection of dUTPase in intracranial tumors.

The nuclear isoform of deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, OMIM *601266, EC 3.6.1.23) is immunohistochemically detectable in all proliferating tissues and may thus be a useful adjunct for the grading of tumors analogous to Ki-67 labeling. A hundred and twenty-seven human intracranial tumors, including 56 astrocytomas, 12 oligodendrogliomas, 8 oligoastrocytomas, 34 meningiomas, 7 ependymomas, and 10 metastatic carcinomas, were stained using the monoclonal rat anti-human dUTPase antibody (clone 3E6) with formalin-fixed and paraffin-embedded tissue. The labeling indices were compared with those obtained with the proliferation marker Ki-67 on parallel tissue sections. All tumors contained dUTPase-positive nuclei, whereas the percentage of positive tumor cells generally increased with grade of malignancy. Meningiomas of higher grades, i.e., World Health Organization (WHO) grades II and III, contained additional cells with cytoplasmic reactivity. There were usually fewer dUTPase- than Ki-67-positive nuclei detectable. Unlike Ki-67, dUTPase was not detectable in mitotic figures. Labeling indices for dUTPase, but not for Ki-67, showed significant differences between all 3 WHO grades of diffuse astrocytomas. In summary, dUTPase staining provides a useful measure of cell proliferation distinct from that offered by Ki-67 labeling. It proved particularly useful for the evaluation of diffuse astrocytomas.

Evidence of focal genetic microheterogeneity in glioblastoma multiforme by area-specific CGH on microdissected tumor cells.

The term "multiforme" in glioblastoma multiforme (GBM) indicates the highly variable histomorphology that cannot be addressed by studies on homogenized tissue probes. In order to relate genetic findings with histomorphologically distinct areas we used microdissection to procure defined cell populations from microscopic tissue sections under direct visualization. Formalin-fixed and paraffin-embedded tissue sections of 10 GBM were evaluated for intratumoral genetic heterogeneity by microdissection of multiple areas of 20-50 tumor cells and DOP-PCR of DNA isolated from the dissected cell groups, followed by comparative genomic hybridization (CGH). Microdissected cells from histomorphologically normal extratumoral blood vessels from the same slides served as controls. The individual tumors showed variable combinations of primary chromosomal gains and losses common to all studied areas of a given case along with secondary, area-specific additional aberrations. CGH displayed a wider variety of chromosomal aberrations than metaphase cytogenetics of cell cultures from the same tumors. The most frequent aberrations observed were previously unperceived gains on chromosomes 4q (8/10) and 5q (5/10). Other nonrandom aberrations were gains on 12q (6/10), 13q (6/10), and 7 (5/10), and losses of 22 (5/10). Amplifications on 7p were intratumorally heterogeneous and only found in single areas of 2 tumors. In contrast to normal extratumoral vessels, vascular proliferates in most cases demonstrated chromosomal aberrations (CGH) which were partially different from the aberrations observed in the tumor itself. The described method gives evidence of considerable intratumoral genetic heterogeneity in GBM and provides a sensitive tool for the detection of quantitative chromosomal changes that are present only regionally within a given tumor.

Case of the month: May 1997--a 31 year old woman with seizures, fatigue and attention deficits.

A 31-year-old woman with a five year history of seizures complained of worsening fatigue and attention deficits. At age 26, neuroradiological imaging studies had shown a small, non-enhancing hypodense lesion involving the left frontobasal and subcallosal region. Follow-up CT and MRI images showed that the lesion had not changed. A stereotactic biopsy showed a dysembryoplastic neuroepithelial tumor (DNT). The lesion was subtotally resected and the diagnosis of DNT was fully confirmed. However, the distinction between oligodendroglioma, ganglioglioma, focal dysplastic cerebral cortex, and DNT was only possible with knowledge of the clinical and neuroradiological data.

An unusual subdural empyema: case report.

Subdural empyema in a 38-year-old patient with congenital hemangioma, suppurative parotitisis, soft tissue phlegmonia and osteomyelitis is reported. The clinical, radiological and surgical features are outlined. A review of the literature reveals the uniqueness of this case.

Distribution of epidermal growth factor receptor protein correlates with gain in chromosome 7 revealed by comparative genomic hybridization after microdissection in glioblastoma multiforme.

In a recent study, 23 microdissected areas of 10 glioblastoma multiforme (GBM) were investigated for quantitative genomic aberrations using comparative genomic hybridization (CGH). To validate the chromosomal aberrations, as revealed by CGH after microdissection, parallel tissue sections were stained immunohistochemically with an antibody that detects both wild-type epidermal growth factor receptor (EGFR) and the deletion mutant form of the receptor (EGFRvIII). Immunostaining was correlated with CGH data of chromosome 7, because chromosome 7 is the most frequently aberrant chromosome in GBM (here four of 10 tumors), and this aberration often indicates an abnormality of EGFR. Nine of nine areas that showed gain in or amplification (2 areas) of chromosome 7 with CGH contained EGFR-immunoreactive cells. Only three of 14 areas without abnormality of chromosome 7 in CGH contained EGFR-immunoreactive cells; eleven of 14 areas were immunonegative. Our findings demonstrate a strong correlation between immunohistochemistry of EGFR and the copy numbers of chromosome 7, as revealed by CGH after microdissection in glioblastoma multiforme.

Initial evaluation of the feasibility of single photon emission tomography with p-[123 I]iodo-L-phenylalanine for routine brain tumour imaging.

p-[123I]iodo-L-phenylalanine (IPA) is a recently described radiopharmaceutical which is highly accumulated in gliomas. The present investigation was designed to evaluate the feasibility of single photon emission tomography (SPET) with IPA to image brain tumours under routine clinical conditions. Using a dual- and a triple-headed SPET camera, whole-body kinetic and brain SPET, as well as plasma, urinary and dosimetric analysis were determined in four patients with gliomas after intravenous injection of IPA. Results obtained by IPA SPET were retrospectively compared with histopathology, magnetic resonance imaging and positron emission tomography with [18F]fluorodeoxyglucose. Tumour lesions were clearly demonstrated by IPA SPET at 30 min, 1h and 4.5h post-injection, even in patients with low grade gliomas. In patients with glioblastoma, excellent visualization of the tumour was possible even at 7h p.i., indicative of the high retention of the radiopharmaceutical in cerebral gliomas. Analysis of the radioactivity in plasma and urine attested to the high in vivo stability of IPA. Blood clearance was rapid (> 65% after 10 min) and IPA was excreted predominantly by the kidneys, the urinary radioactivity excretion ranging from 27% at 1h to 54% of injected doses at 5h p.i. The average effective dose for adults was estimated to be 0.0152mSv*MBq(-1), leading to an effective dose of 3.8mSv in a typical brain SPET investigation with 250 MBq IPA. This result strongly suggests that IPA is a potentially valuable brain tumour imaging agent for widespread clinical studies with SPET. Its high specific tumour uptake and retention even in low grade gliomas represent a major advantage compared to presently available SPET radiopharmaceuticals. Moreover, the radiation dose estimates indicate that clinical use of IPA will result in acceptable radiation dose levels in humans.

Demonstration of platinum microcoils in embolized blood vessels in-situ using a modified methyl methacrylate embedding method, and a special cutting and grinding technique.

With current paraffin embedding techniques it is not possible to demonstrate metal particles in vascular lumens. We used a modified methyl methacrylate embedding method for obtaining plastic blocks which were cut with a precision diamond saw, and subsequently ground and polished to preserve the interface of reactive tissue surrounding platinum microcoils in arteriovenous malformations. Artifact-free interfaces could be achieved by slow embedding under vacuum conditions, slow polymerization in a water bath, and very gentle cutting, grinding and polishing techniques. The method produces good preservation of tissue and cells directly adjacent to metal coils. Our method is useful for the histomorphological interpretation of vascular pathologies in which metal particles have been introduced.

Long-term results with Matrix coils vs. GDC: an angiographic and histopathological comparison.

INTRODUCTION: The aim of the study was to compare standard platinum Guglielmi detachable coils (GDC) with coated platinum coils (Matrix; both Boston Scientific, Fremont, CA) regarding handling, complications, occlusion and recanalization rate after 3 and 6A months. METHODS: Aneurysms in the right common carotid artery were created in 25 rabbits. The animals were divided into five groups of five animals each. The animals of group 1 (the control group) received no treatment of the induced aneurysms, the animals of groups 2 and 3 (killed at 3 and 6A months) were treated with standard GDC, and the animals of groups 4 and 5 (killed at 3 and 6A months) were treated with Matrix coils. RESULTS: Histopathological evaluation showed organized thrombus formation and connective tissue with neovascularization around the implanted coils in all the treated groups. The achieved occlusion rates in groups 2 and 3 were identical to those in groups 4 and 5. Thus the long-term results of aneurysm treatment with GDC and Matrix coils show no differences regarding occlusion and recanalization rates. The only noticeable difference was the difference in handling. More force was required to pushing the Matrix coils forward through the microcatheter and there was more friction in coil interaction in the aneurysm. CONCLUSION: The bioactive coating of the Matrix coil produces no significant benefit in achieving higher occlusion and lower recanalization rates, and the coil is more difficult to handle. Future bioactive coils must be shown to produce significantly better long-term results than GDC and their ease of handling has to be improved.